Nucleic Acids Research, Vol 25, Issue 19 3925-3929, Copyright © 1997 by Oxford University Press
BB Rosenblum, F Oaks, S Menchen and B Johnson
Interpolation algorithms can be developed to size unknown single- stranded
(ss) DNA fragments based on their electrophoretic mobilities, when they are
compared with the mobilities of standard fragments of known sizes; however,
sequence-specific anomalous electrophoretic migration can affect the
accuracy and precision of the called sizes of the fragments. We used the
anomalous migration of ssDNA fragments to optimize denaturation conditions
for capillary electrophoresis. The capillary electrophoretic system uses a
refillable polymer that both coats the capillary wall to suppress
electro-osmotic flow and acts as the sieving matrix. The addition of 8 M
urea to the polymer solution, as in slab gel electrophoresis, is
insufficient to fully denature some anomalously migrating ssDNA fragments
in this capillary electrophoresis system. The sizing accuracy of these
fragments is significantly improved by the addition of 2-pyrrolidinone, or
increased capillary temperature (60 degrees C). the effect of these two
denaturing strategies is additive, and the best accuracy and precision in
sizing results are obtained with a combination of chemical and thermal
denaturation.
ARTICLES
Improved single-strand DNA sizing accuracy in capillary electrophoresis
PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. rosenbbb@perkin-elmer.com
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